We found that progenitor cells persisted at later time points, indicating that extending time in culture may be of limited use for eliminating them

We found that progenitor cells persisted at later time points, indicating that extending time in culture may be of limited use for eliminating them. from stem cells. They further undertake single-cell and reporter gene approaches to select highly differentiated neurons with increased functionality, augmenting their utility in the Rabbit Polyclonal to 14-3-3 beta modeling of nervous system disorders. INTRODUCTION Progress toward producing more accurate models of human brain cell types continues to be made (Brennand et al., 2015; Pa?ca et al., 2015). Directed differentiation approaches aim to mimic embryonic development by stepwise specification of neuronal subtypes (Chambers et al., 2009; Espuny-Camacho et al., 2013; Zhang et al., 2013; Ho et al., 2015). In one such strategy, pluripotent stem cells (PSCs) can be neuralized through the inhibition of bone morphogenetic protein (BMP) and transforming growth factor (TGF-) signaling (Chambers et al., 2009; Maroof et al., 2013), regionally specified with morphogens, and then allowed to differentiate. While this approach enables cells to transit through cellular states normally observed during embryogenesis, differentiation unfolds slowly. Generation of early post-mitotic forebrain neurons can take as long as 5 weeks, while the production of astrocytes or oligodendrocytes requires even more extended times in culture (Tao and Zhang, 2016). In contrast, transcription factor-programming approaches rely on Exatecan mesylate ectopic expression of lineage-specific transcription factor(s), in either somatic cells or PSCs, to achieve a rapid cell fate conversion (Son et al., 2011; Mertens et al., 2016). It has been shown that Ascl1, Brn2, and Myt1l can convert mouse fibroblasts into induced neurons (iNs) in as little as 2 weeks (Vierbuchen et al., 2010). More recently, expression of the neuralizing transcription factor NGN2 in human PSCs (hPSCs) was reported to induce an excitatory neuronal identity in a similar time frame (Zhang et al., 2013). While these methods allow more rapid production of human neurons, insight into the heterogeneity of differentiated neurons remains limited. Indeed, using single-cell analysis, it was revealed that, in addition to producing iNs, Exatecan mesylate expression has routinely been observed only at very late stages of differentiation (up to 145 days in culture) (Gupta et al., 2013; Kirwan et al., 2015). Generation of stem cell-derived Exatecan mesylate neurons with robust NMDAR-mediated synaptic transmission would have specific translational value, as variants in and around the glutamate ionotropic receptor NMDA type subunits 2A and 2B (and led to more effective neutralization, resulting in cells that expressed transcription factors expressed in superficial levels of the cortex. Although these cultures were homogenously neuralized, cells existed in transcriptional states that ranged from early progenitor to well-differentiated excitatory neuron states. More differentiated cells expressing and subunits also expressed reporter gene. This approach allowed the isolation of highly differentiated and synaptically active human patterned induced neurons (hpiNs), underscoring the potential utility of this approach for modeling diseases associated with glutamate receptor dysfunction, including schizophrenia, epilepsy, and autism (Yamamoto et al., 2015; Yuan et al., 2015). RESULTS Patterning of NGN2-Induced hPSCs with Dual SMAD and WNT Inhibition Previously, it has been shown that forced expression of the NGN2 transcription factor in hPSCs can induce rapid differentiation into cells Exatecan mesylate with excitable membranes and capable of synaptic function (Zhang et al., 2013). We set out to investigate whether the extrinsic influences of small molecules that inhibit BMP and TGF- signaling (Chambers et al., 2009; Maroof et al., 2013) could favorably synergize with the activities of NGN2 (Figure 1). To this end, NGN2 expression was induced in TetO-NGN2-T2A-PURO/TetO-GFP lentivirally infected human stem cells by exposure to doxycycline (dox) 1 day after plating. To induce patterning toward a forebrain phenotype, cells were neuralized Exatecan mesylate by inhibiting TGF- and BMP signaling (treatment with SB431542 and LDN193189), and they were dorsalized by inhibiting Wnt signaling (treatment with XAV939, a tankyrase inhibitor) for 3 days. Puromycin was then applied to select for cells expressing NGN2. The differentiation scheme was performed on both hESC (human embryonic stem cell) and hiPSC lines generated from fibroblasts of healthy individuals (iPS1 and iPS2). At 4 days post-dox induction (day 4), cells were co-cultured with mouse astrocytes to promote neuronal maturation and synaptic connectivity (Pfrieger, 2009; Eroglu and Barres, 2010). Consistent with previous observations (Zhang et al., 2013), changes in cell.